Dissertation / PhD Thesis/Book

PreJuSER-13576

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Untersuchungen von $\textit{in vivo}$-Wechselwirkungen zwischen sekretorischen Vorläuferproteinen und Komponenten des Proteinexportapparates Gram-positiver Bakterien

Tippe, D. (Corresponding author)FZJ*

2001
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich

Please use a persistent id in citations: http://hdl.handle.net/2128/19980

Report No.: Juel-3877

Abstract: Trials to secrete heterologous proteins using Gram-positive bacteria as host organisms have shown problems with regard to the translocation step across the cytoplasmic membrane. The reason for this are presumably non optimal interactions between the subunits of the translocase (Sec-components) and the precursor proteins. In order to analyse the interactions between Seccomponents and heterologous precursor proteins, $\textit{Bacillus subtilis / Staphylococcus carnosus}$ hybrid translocases were used. The starting point of this thesis was a secA-replacement mutant of $\textit{B. subtilis}$ (RMA). In this mutant, the $\textit{B. subtilis}$ secA gene is chromosomally replaced by the secA of $\textit{S. carnosus}$. The gene replacement results in disturbed interactions between the $\textit{S. carnosus}$ SecA protein (SecA(Sc)) and at least one translocase subunit of $\textit{B. subtilis}$ (SecX(Bs)). As a result of this perturbation the RMA-mutant shows a conditional protein export defect leading to coldsensitive growth . In addition a heterologous protein (pre-OmpA of $\textit{Escherichia coli}$) is selectively discriminated from protein export. The aim of this thesis was to identify the disturbing SecA(Sc)- SecX(Bs) interactions in the RMA. To reach this goal additional translocase subunits (SecX(Sc)) of S. carnosus should be specifically introduced into the RMA-mutant. For the project it was necessary to clone the missing $\textit{S. carnosus}$ sect gene. Using the $\textit{B. subtilis}$ secG gene as probe a hybridizing DNA-fragment of $\textit{S. carnosus}$ was identified and subsequently cloned. Sequence analysis revealed that this fragment encodes a protein showing homology to the SecG proteins of $\textit{B. subtilis}$ and $\textit{E. coli}$. The protein characterization revealed that it is necessary for an efficient protein export $\textit{in vivo}$ and $\textit{in vitro}$. The characterisation of a secG replacement mutant of $\textit{B. subtilis}$ (RMG), in which the $\textit{B. subtilis}$ secG gene is chromosomally replaced by secG of $\textit{S. carnosus}$ showed that the $\textit{S. carnosus}$ SecG protein optimally interacts with the remaining translocase subunits of $\textit{B. subtilis}$. The RMG behaves as the $\textit{B. subtilis}$ wildtype in contrast to the above mentioned RMA-mutant, in which an interaction between the $\textit{S. carnosus}$ SecA and a so far unknown $\textit{B. subtilis}$ Sec-component is disturbed. Additional replacement of the $\textit{B. subtilis}$ secG-gene on the chromosome of the RMA by the corresponding gene of $\textit{S. carnosus}$ (RMA/G) did not change the RMA phenotypes. The replacement mutant RMA/G behaved like the RMA. This result and the behaviour of the RMG clearly show that the SecA(Sc)-SecG(Bs)-interaction in the RMA is functional. In contrast to the $\textit{S. carnosus}$ SecG, introduction of the $\textit{S. carnosus}$ SecY protein into the RMA abolished the cold sensitive phenotype and the selective discrimination of the heterologous OmpA precursor from protein export. This clearly shows that the SecA(Sc)-SecY(Bs)-interaction is disturbed in the RMA. In addition, these results indicate that the pre-OmpA (as probably all secretory proteins) interacts simultaneously with SecA and SecY at an early step of protein translocation. Hence, the precusor is an equivalent part of an at least trimeric complex formed by precursor, SecA, and SecY. Apart from this it was shown in comparable investigations that besides an optimal SecA-SecY-interaction, an optimal interaction between the SecE and the SecY protein is also important for an efficient protein translocation. The results of this thesis suggest, that the often observed, inefficient or even entirely prevented membrane translocation of heterologous proteins using Gram-positive bacteria as hosts is due to an inefficient or unproductive formation of a translocation initiation complex of precursor, SecA, SecY, and (most likely) SecE.

Note: Record converted from VDB: 12.11.2012
Note: Düsseldorf, Univ., Diss., 2001

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Contributing Institute(s):

1. Biotechnologie 1 (IBT-1)

Research Program(s):

1. Mikrobiologische Grundlagen der Gewinnung von Proteinen (41.35.1)

Appears in the scientific report 2001

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Database coverage:

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